Pleiotropic effects of the GREY mutation in horses

In horses, premature greying of the coat is caused by 4.6 kb duplication in intron 6 of STX17 that causes overexpression of STX17 and neighbouring genes (e.g. NR4A3). This fully dominant mutation (GREY mutation) was discovered by Leif Andersson (Uppsala University). In Vienna, Johann Sölkner (BOKU) with partners from VUW, Medical University of Vienna (MUW) and University of Zagreb had investigated the genetics of greying, melanoma and a vitiligo syndrome in GREY Lipizzaner horses of European state studs (Austria, Slovenia, Croatia, Hungary, Bosnia Herzegovina, Italy and Slovakia) by several repeated phenotyping and collection of pedigree information over several years. Based on this, both research groups investigated the effects of genotype (mutation) on phenotypes (speed of greying, melanoma, vitiligo and speckling/freckles). The results indicate that homozygous GREY horses are greying faster and are more prone to melanoma and vitiligo than heterozygous animals whereas almost all speckled horses are heterozygous (Rosengren Pielberg et al. 2008). This work received very much attention all over the world. The primary goal of the current project is to fully understand the (molecular) mechanism behind this GREY mutation, its function, impact (CNV) and effects on premature greying, on melanoma development and susceptibility for tumorigenesis, on development of vitiligo, speckling (freckles) of coat and skin, as well on melanocyte cell migration. We screen for changes in gene expression associated with the presence of this mutation by means of full transcriptome analysis in melanocytes from GREY versus non-GREY horses using next-generation sequencing (NGS), scRNA-seq, copy number analysis (CNV), and CRISPR/Cas9 as well as TALEN-mediated knockout experiments. Additionally, proteome analysis of all cell types obtained in the frame of this study (melanocyte-stem- cells, different melanocytes out of distinct skin areas, primary melanoma cells) but also appropriate tissue specimens should allow us to gain a deeper insight into the functional and regulatory mechanisms behind the GREY mutation.